Project Summary Despite evidence of strong genetic contributions to the etiology of nicotine dependence (ND), we are far from identifying the specific genetic bases of individual susceptibility to ND. The primary objective of this proposal is to identify novel genetic factors that contribute to nicotine withdrawal, an important aspect of ND that contribute relapse, in mice. We observed pronounced nicotine withdrawal traits differences in C57BL/6NJ strain but not in the closely related C57BL/6J substrain. Because the parental substrains are nearly genetically identical, quantitative trait locus (QTL) mapping in an experimental F2 cross (Reduced Complexity Cross; RCC) will greatly facilitate the identification of novel genetic factors that underlie differences in withdrawal behaviors. In Aim 1, we will use the RCC to map genomic regions, or QTLs, that are causally associated with susceptibility versus resilience to multiple measures of nicotine withdrawal. In Aim 2, we will conduct transcriptome analysis via mRNA sequencing (RNA-seq) of four brain regions regions in control mice and chronic nicotine-treated mice from the parental male and female C57BL/6J and C57BL/6NJ substrains. The transcriptome in control mice will serve as a useful tool both in identifying candidate genes for future genome editing that are differentially expressed and underlie the behavioral QTLs as well as providing genomic insight into the neuronal context that influences susceptibility versus resilience to nicotine withdrawal. Genes that are differentially expressed as a consequence of nicotine will reveal changes in the transcriptome relevant to central neuronal plasticity and the behaviors/changes that support ND. In Aim 3, we will validate candidate quantitative trait genes and functional variants identified and ranked by Aims 1-2.